IKK2/NFkB signaling controls lung resident CD8+ T cell memory during influenza infection

CD8+ T cell tissue resident memory (TRM) cells are especially suited to control pathogen spread at mucosal sites. However, their maintenance in lung is short-lived. TCR-dependent NFkB signaling is crucial for T cell memory but how and when NFkB signaling modulates tissue resident and circulating T cell memory during the immune response is unknown. Here, we find that enhancing NFkB signaling in T cells once memory to influenza is established, increases pro-survival Bcl-2 and CD122 levels thus boosting lung CD8+ TRM maintenance. By contrast, enhancing NFkB signals during the contraction phase of the response leads to a defect in CD8+ TRM differentiation without impairing recirculating memory subsets. Specifically, inducible activation of NFkB via constitutive active IKK2 or TNF interferes with TGFβ signaling, resulting in defects of lung CD8+ TRM imprinting molecules CD69, CD103, Runx3 and Eomes. Conversely, inhibiting NFkB signals not only recovers but improves the transcriptional signature and generation of lung CD8+ TRM. Thus, NFkB signaling is a critical regulator of tissue resident memory, whose levels can be tuned at specific times during infection to boost lung CD8+ TRM.


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Raw data generated in this study have been deposited in the public Open Science Framework (OSF) https://osf.io/2vtur/, with no accession code needed. Raw flow files are available upon request due to size restrictions. Access can be obtained by contacting the corresponding author. Uncropped, unprocessed scans of the most important blots appeared in supplementary information Figures 13 and 14. The authors declare that the data supporting the findings in this study are available within the paper and its supplementary information files. Source data are provided with this article.
Power calculations were performed in Graph Pad Prims for multiple or two-tailed unpaired students' t-tests, using variance and effect size necessary to achieve sufficient N to determine p-values. A minimum of n=2 was chosen because it was the minimal replicate number sufficient to ascertain statistcs by t-test.
No data were excluded except from data from mouse replicates where an error in the infection dose or in the staining was identified during the experimental procedure. It should be noted that the observed differences were statistically significant whether these data points were included or not.
All experiments were independently conducted a minimum or two times to ensure reproducibility. The number of biological replicates (mice) per cohort or condition for each independent experiment is noted in each Figure legend. Immunoblot analysis were repeated at least two times with completely independent biological replicates and using two different CA-IKK2ON inducible models. Biological replicates for immunoblot experiments have been noted in the pertinent figure legends.
Mice were randomly assigned to each study group Blinding was performed for immunohistochemistry experiments and analysis. Blinding was not performed for other data for biosafety concerns associated with BSL2 samples.

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April 2023 All antibodies were validated by the manufacturers following consistent performance and specific binding activity (BD, Biolegend, Rockland, Cell Signaling, R&D, ThermoScientific and Sigma ). Details can be found in the manufacturer's websites by referring to the catalague number of the antibodies above. In addition, specific detection was confirmed by us: (1) by ensuring lack of signal with isotype controls conjugated with same fluorochrome or (2) by lack of signal upon staining of the specific antibody in control populations that do not express the antigen. Please see Fig. 5b Note that full information on the approval of the study protocol must also be provided in the manuscript.

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Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots. NO wild animals were used.
Female and male mice were not disaggregated as no gender bias was found. Experiments in Fig. 3 required designated genders otherwise, experiments were gender matched following similar studies using the same approach that were published in the peer reviewed Journal Nature 2011 (specific citation reference in main manuscript).

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All procedures were conducted according to the NIH guidelines for the care and use of laboratory animals and biological safety and were approved by the University of Missouri Institutional Animal Care and use and Institutional Biosafety Committees.
General FACs immunostatining. Lungs, kidney, spleen, and mediastinal lymph node tissues were harvested, and lymphocytes isolated. Next, lymphocytes were stained in vitro with anti-CD8! antibodies along with fluorochrome conjugated antibodies specific of other surface markers resuspended in FACS buffer (PBS/1% fetal bovine serum). For immunostaining of intracellular markers such as transcription factors T-bet, Eomes or Nur77, after staining of cell surface markers, cell samples were rinsed, fixed and permeabilized with Cytofix/cytoperm. Stained cells were run on a LSR Fortessa flow cytometer (BD, San Jose, CA, data collected with FACS Diva 6.0 by BD) or run on a Cytek Aurora spectral flow cytometer using the Cytek Spectraflo 3.0.3 software. Flow cytometry data was analyzed using with FlowJo software version 10.4 (Tree Star, Inc., Ashland, OR). It should be considered that our observations refer mainly to the generation and maintenance of influenza specific memory CD8 T cells in the lung parenchyma identified through IV labeling, a method widely used to study TRM cells in the field. One limitation of this approach is that it can only provide a snapshot of the cells that are residents in tissue at a given time and it is less accurate at quantifying T cells in transition. A table of antibodies is provided in supplementary information.